Methods for screening GABA-modulatory compounds for specified pharmacological activities

ABSTRACT

Methods are provided that are useful in assaying compounds for cognitive enhancing properties, anxiolytic properties, hypnotic properties, or antidepressant properties. These methods involve determining the in vitro efficacy and EC 50  of the compounds at defined series of cloned GABA A  subtype receptors composed of specific variants of α,β,and γ subunits in order to develop and an activity profile for each compound. Optionally, the binding affinities of the compounds at GABA A  receptors are also determined. As an additional step the in vivo effects of the compounds may be tested in animal models

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisionalapplications No. 60/133,195, entitled “Methods for Screening Compoundsfor Cognition Enhancing Activity”, No. 60/133,191, entitled “Methods forScreening Compounds for Antidepressant Activity”, No. 60/133,155,entitled “Methods for Screening Compounds for Hypnotic Activity”, andNo. 60/133,154, entitled “Methods for Screening Compounds for AnxiolyticActivity”, each of which was filed in the names of the present inventorson May 7, 1999. To the extent that they differ from the disclosure ofthe present application, the disclosures (including the claims) of theseprovisional applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for assayingGABA-modulatory compounds for activity as antidepressants, cognitiveenhancers, sedative hypnotics, or non-sedating anxiolytics. Inparticular, the method includes determining efficacy (generally in vitroefficacy) and EC₅₀ values (as used herein incorporating IC₅₀ values) forthe compounds at several different cloned (i.e., expressed in cells asdirected by heterologous cloned receptor-encoding nucleic acidexpression vector molecules) GABA_(A) subtype receptors (each subtypemade up of a defined set of specific receptor subunit isotypes). Themethod optionally includes determining binding affinity of compounds forGABA_(A) receptors. The method results in the development of an activityprofile for each compound. As an additional step, animal modelspredictive of such effects may be used to measure the ability ofcompounds to effect cognitive enhancement, to act as antidepressants, tomediate sedative hypnotic effects, or to effect anxiolysis in vivowithout eliciting certain undesirable side effects.

[0004] 2. Description of Related Art

[0005] Modern drug discovery methodology allows the testing of largenumbers of compounds (often assembled into collections termed libraries)for functional characteristics that confer pharmaceutical utility. This“screening” of such libraries, using specific tests (assays) forfunctional activity properties, allows the rapid identification ofpromising compounds for further development as pharmaceutical agents.There has been a longstanding quest in the pharmaceutical industry fornew means of identifying such promising compounds. Such new means mayinvolve new assays, or may use old assays to generate data that can beanalyzed and applied in new ways to identify compounds with new anduseful characteristics.

[0006] In the field of psychopharmacology, the use of cloned neuronalreceptors as substrates has provided new, more specific assays withwhich compounds can be characterized. The use of such receptors hasenabled the development of receptor binding profile criteria that areparticularly beneficial in the identification of usefulpsychopharmacological agents. For example, such profiling can identifycompounds that will be free of certain undesirable adverse effects (sideeffects).

[0007] The GABA_(A) receptor superfamily represents one of the classesin of receptors through which the major inhibitory neurotransmitter,γ-aminobutyric acid, or GABA acts. In addition to being the site ofneurotransmitter action a number of drugs including the anxiolytic andsedating benzodiazepines bind to this receptor. The GABA_(A) receptor isa chloride channel that opens in response to GABA, allowing chloride toenter the cell. This effects a slowing of neuronal activity throughhyperpolarization of the cell membrane potential. GABA_(A) receptors arecomposed of several protein subunits and are generally pentameric instructure.

[0008] A number of cDNAs for GABA_(A) receptor subunits have beencloned. While these subunits share a basic motif of 4 membrane-spanninghelices, there is sufficient sequence diversity to classify them intoseveral groups. To date at least 6α, 3β, 3γ, 1ε, 1δ, 2ρ, and 1π subunitspecies have been identified; some representing alternatively splicedforms. Native GABA_(A) receptors are typically composed of α, β, and γsubunits, most often in the ratio of two alphas, two betas, and onegamma, although other combinations (some comprising other subunits suchas ε, δ, ρ, or π) have been described. Even if restricted to only α, β,and γ subunits, however, an enormous diversity of GABA_(A) subtypereceptors are possible. Evidence such as message distribution, genomelocalization and biochemical studies suggests that the major naturallyoccurring receptor combinations are α₁β₂γ₂, α₂β₃γ₂, α₃β₃γ₂, and α₅β₃γ₂.

[0009] In the typical GABA_(A) receptor, the binding sites for GABA (2per receptor complex) are formed by amino acids from the α and βsubunits. Amino acids from the α and γ subunits contribute to form 1benzodiazepine site per receptor complex. In a classic allostericmechanism, the binding of a drug to the benzodiazepine site increasesthe affinity of GABA binding to the receptor. Benzodiazepines andrelated drugs that enhance the ability of GABA to open GABA_(A) receptorchannels are known as agonists or partial agonists depending on thelevel of enhancement. Other classes of drugs such as β-carbolinederivatives that occupy the same site and negatively modulate the actionof GABA are called inverse agonists. A third class of compounds existsthat occupies the same site as both the agonists and inverse agonists(blocking access of these agents to the site) and yet has little or nodirect effect on GABA activity. These compounds are referred to asantagonists.

[0010] The characterization of activities of different subtype receptorshas been an area of intense pharmacological discovery. Agonists that actat the benzodiazepine site have long been known to know to exhibitanxiolytic, sedative, and hypnotic effects in animal behavior models,while compounds that act as inverse agonists at this site elicitanxiogenic, cognition enhancing, and proconvulsant effects. Whilebenzodiazepines have long been used as anxiolytics, these compoundsexhibit a number of undesirable side effects. These include cognitiveimpairment, sedation, ataxia, potentiation of ethanol effects, and atendency for tolerance and drug dependence. Likewise the development ofbenzodiazepine site ligands for other indications has been thwarted byunfavorable side effect profiles for each indication. For example,compounds known to possess cognition enhancing properties have generallytended to be anxiogenic and proconvulsant, while compounds that produceanxiolytic effects tend to generate unwanted sedation, and do so morepowerfully when taken in conjunction with the consumption of alcoholicbeverages.

SUMMARY OF THE INVENTION

[0011] The present invention provides methods for characterizingcompounds that act at the GABA_(A) receptor benzodiazepine site. Inparticular, it provides methods for identifying compounds withcharacteristics indicating that the identified compounds will exhibitpharmacological properties consistent with their use as antidepressants,cognitive enhancers without anxiogenic or proconvulsant activity,sedative hypnotics without cognition-impairing activity, or non-sedatinganxiolytics.

[0012] This invention is useful in screening libraries of compounds fortherapeutic potential and in drug design efforts.

[0013] Measurement of GABA receptor binding affinity is a useful step inany of the methods of the invention. Alternatively, these assays may beperformed without measuring the binding affinity of the compound. Theassays may include an assessment of the ability of the compound tomediate the desired effects in vivo without eliciting side effects usinganimal models established to be predictive of the desired effects andanimal models predictive of the undesired side effects that have beenassociated with other compounds acting at GAB_(A) receptors.

[0014] As used herein, the term “efficacy” refers to amount ofpotentiation (represented as a % increase, e.g., 10%) or inhibition(represented as a % decrease, e.g., −10%) of GABA activated responsesmeasured for GABA_(A) receptors.

[0015] In addition to the ability of a compound to effect a specifiedmagnitude of change in the GABA response at distinct subtype receptors,the EC₅₀ value of the compound at the pertinent receptors is also takeninto consideration. As used herein, the term “EC₅₀” or “EC₅₀ value”refers to the concentration of a compound needed to elicit half themaximal response (to the agonist or inverse agonist effects of acompound) that can be obtained with the compound. Thus, a compound thatexhibits dissimilar EC₅₀ values at different subtype receptors canselectively potentiate one of those receptors over a defined range ofdrug concentrations, even though the maximal amount of potentiationachievable by the compound is the same for the two subtype receptorsover a much broader range of compound concentrations. EC₅₀ values do notnecessarily correlate to binding affinities or to compound efficacies.

[0016] Cognitive Enhancers: With regard to identifying cognitiveenhancers, a method of the invention involves optionally determining thebinding affinity of a compound for GABA_(A) receptors having Ro15-1788binding sites and determining efficacy and EC₅₀ values for the compoundat cloned α₁β₂γ₂ and α₅β₃γ₂ receptors and comparing these values withefficacy and EC₅₀ values for the compound at cloned GABA_(A) receptorscontaining the α₂ or α₃ subunits. The ability of the compound to mediatecognitive enhancing effects may optionally be assessed in vivo by animalmodel predictive of cognitive enhancement. Whether the compound causesproconvulsant effects may also be assessed in vivo using animal modelsfor detecting proconvulsant activity.

[0017] Accordingly, in one aspect this invention provides methods foridentifying compounds with cognitive enhancing activity that do notdisplay the side effects of anxiogenesis or proconvulsant activity.These methods comprise:

[0018] a) Screening compounds, optionally determining the bindingaffinity of the compounds for GABA_(A) receptors;

[0019] b) determining in vitro efficacy and EC₅₀ values of the compoundsusing cloned α₁β₂γ₂ and α₅β₃γ₂ receptors and comparing these values toin vitro efficacy and EC₅₀ values for the compound determined usingGABA_(A) receptors that contain the α₂ or α₃ subunit; and

[0020] c) selecting compounds having significant inverse agonistcharacter and sufficiently low EC₅₀ values at α₁β₂γ₂ or α₅β₃γ₂ subtypereceptors and that produce agonist activity at GABA_(A) subtypereceptors that contain the α₂ or α₃ subunit.

[0021] Thus, the invention presents novel methods for identifyingcompounds with selective cognitive enhancing properties (1) by examiningthe binding of a given compound at GABA_(A) receptors and (2) byassessing the ability of the compound to potentiate GABA responses at aseries of GABA_(A) subtype receptors. These values are then compared toa set of criteria termed the “Cognitive Enhancer Activity RangeProfiler” (Table I, below). This activity profile comprises measurementsof in vitro efficacy (agonist, inverse agonist or antagonist character)and EC5₀ values at each of 4 GABA_(A) subtype receptors. The activityprofile needed for cognitive enhancement is presented as a precisewindow of inverse agonism at certain subtype receptors and agonism atother subtype receptors. The EC₅₀ criteria at each of these subtypereceptors are also presented. Determining efficacy and EC₅₀ values for atest compound is crucial as many compounds bind with high affinity atthe benzodiazepine site without potentiating the GABA response at theappropriate subtype receptors.

[0022] In certain embodiments, the conclusions drawn from the in vitrodeterminations of validity may be confirmed by examining the in vivoefficacy of test compounds as cognitive enhancers using animal modelsfor cognitive enhancement.

[0023] It may also be necessary to verify that compounds identified bythese methods possess the predicted favorable side effect profiles byexamining the performance of the compounds in animal models indicativeof these side effects. Thus, the animal models may be used as anadditional step of the assay to further refine the selection ofcompounds with cognitive enhancing activity.

[0024] Anxiolytics: With regard to identifying anxiolytics, a method ofthe invention involves optionally measuring the binding affinity of acompound at GABA_(A) receptors having Ro15-1788 binding sites andmeasuring the efficacy and EC₅₀ values for a compound using clonedα₂β₃γ₂ and α₃β₃γ₂ receptors and comparing these values with theactivities and EC₅₀ values of the compound at cloned GABA_(A) receptorscontaining the α₁ or α₅ subunits. As an additional step, the ability ofthe compound to mediate anxiolytic effects may be assessed in vivo usingan animal model established to be predictive of anxiety, and whether thecompound causes sedative effects may also be assessed in vivo by ananimal model shown to measure sedation.

[0025] Accordingly, this invention provides a method for identifyingcompounds with anxiolytic activity that do not display the side effectsof cognitive impairment, ataxia, potentiation of alcohol effects, and atendency for tolerance and drug dependence or that display these sideeffects only to a very minimal degree. These method comprise:

[0026] a) screening compounds, optionally measuring the binding affinityof the compounds at GABA_(A) receptors;

[0027] b) measuring the in vitro efficacy and EC₅₀ values of thecompounds at cloned α₃β₃γ₂ and α₃β₃γ₂ receptors and comparing thesevalues to the in vitro efficacy and EC₅₀ values of the compounds atGABA_(A) receptors that contain the α₁ or α₅ subunit; and

[0028] c) selecting compounds with partial agonist character andsufficiently low EC₅₀ values at α₂β₃γ₂ and α₃β₃γ₂ subtype receptors thatalso display lower activity at GABA_(A) subtype receptors that containthe α₁ or α₅ subunit.

[0029] Alternatively, this assay may be performed without measuring thebinding affinity of the compound or with the additional step ofassessing the ability of the compound to mediate anxiolytic effects invivo without causing sedation via an animal model established to bepredictive of anxiety and an animal model predictive of sedativeeffects.

[0030] Thus, the invention presents a method for identifying compoundswith selective anxiolytic activity (1) by examining the binding of agiven compound at GABA_(A) receptors and (2) by assessing the ability ofthe compound to potentiate GABA responses at a series of GABA_(A)subtype receptors. The resulting values are then compared to a set ofempirically defined criteria termed the “Anxiolytic Activity RangeProfiler” (Table III, below). The criteria given by the AnxiolyticActivity Range Profiler are used to select compounds with anxiolyticactivity that have no or very minimal sedative effects. This activityprofile comprises determinations of in vitro efficacy (agonist, inverseagonist or antagonist character) and EC₅₀ values at each of 4 GABA_(A)subtype receptors. The activity profile needed for anxiolysis ispresented as a precise window of agonism and EC₅₀ criteria at each ofthese subtype receptors. The combination of determining efficacy, andEC₅₀ values for a test compound is crucial as many compounds bind withhigh affinity at the benzodiazepine site without potentiating the GABAresponse at the appropriate subtype receptors.

[0031] In certain embodiments, the conclusions drawn from the in vitrodeterminations may be confirmed by examining the in vivo efficacy oftest compounds predicted to have anxiolytic activity in an animal modelfor anxiety. It may also be desirable to verify that compoundsidentified by these methods possess the predicted favorable side effectprofiles by examining the performance of the compounds in animal modelsknown to be indicative of sedative effects. Thus, the animal models maybe used as an additional step of the assay to further refine theselection of compounds with non-sedating anxiolytic properties.

[0032] Antidepressants: With regard to identifying antidepressants, amethod of the invention involves optionally measuring the bindingaffinity of a compound at GABA_(A) receptors, measuring the efficacy andEC₅₀ values for a compound at cloned α₂β₃γ₂ and α₃β₃γ₂ receptors, andcomparing these values with efficacy and EC₅₀ values for the compound atcloned GABA_(A) receptors containing the α₁ or α₅ subunits. Optionally,the ability of the compound to mediate antidepressant effects may beassessed in vivo using one or more animal models predictive ofantidepressant activity. Whether a compound produces sedative effectsmay also be assessed in vivo with an animal model for measuringsedation.

[0033] Accordingly in a broad aspect, this invention provides a methodfor screening compounds for antidepressant activity that do not causethe side effects of cognitive impairment, ataxia, potentiation ofalcohol effects, and a tendency for tolerance and drug dependence, orthat display these side effects only at a very low level. These methodcomprise:

[0034] a) screening compounds, optionally measuring the binding affinityof the compounds at GABA_(A) receptors;

[0035] b) determining in vitro efficacy and EC₅₀ values for thecompounds at cloned α₂β₃γ₂ and α₃β₃γ₂ receptors and comparing thesevalues to in vitro efficacy and EC₅₀ values for the compounds determinedat GABA_(A) receptors that contain an α₁ or α₅ subunit; and

[0036] c) selecting compounds having partial agonist character and thatproduce sufficiently low EC₅₀ values at α₂β₃γ₂ and α₃β₃γ₂ subtypereceptors and display lower efficacy activity at GABA_(A) subtypereceptors that contain the α₁ or α₅ subunit.

[0037] The compounds selected by this method have antidepressantactivity.

[0038] The assay may include an assessment of the ability of thecompound to mediate antidepressant effects in vivo without causingsedation. Animal models predictive of antidepressant effects andsedative effects can be used for the in vivo determination.

[0039] Thus, the invention presents methods for identifying compoundswith antidepressant activity, i.e., selective antidepressant activity,(1) by examining the binding of a given compound at GABA_(A) receptorsand (2) by assessing the ability of the compound to potentiate GABAresponses at a series of GABA_(A) subtype receptors. The resultingvalues are then compared to a set of criteria termed the “AntidepressantActivity Range Profiler” (Table IV, below). The criteria given by theAntidepressant Activity Rang Profiler are used to identify compoundswith antidepressant activity that have no or very minimal sedativeeffects. This activity profile comprises determinations of in vitroefficacy (agonist, inverse agonist or antagonist character) and EC₅₀values at each of 4 GABA_(A) subtype receptors. The activity profileneeded for antidepressant activity is presented as a precise window ofagonism and EC₅₀ values at each of these subtype receptors. Thecombination of determining efficacy and EC₅₀ values for a test compoundis crucial as many compounds bind with high affinity at thebenzodiazepine site without potentiating the GABA response at theappropriate subtype receptors. Furthermore, as noted previously, EC₅₀values do not necessarily correlate with binding affinities or compoundefficacies.

[0040] In certain embodiments, the conclusions drawn from the in vitrodeterminations may be confirmed by examining the in vivo effects of testcompounds selected as having antidepressant activity using animal modelsfor depression. It may also be desirable to verify that compoundsidentified by these methods do indeed possess the predicted favorableside effect profiles by examining the performance of the compounds inanimal models known to be indicative of these side effects. Thus, theanimal models may be used as an additional step of the assay to furtherrefine the selection of compounds with selective antidepressantactivity.

[0041] Hypnotics: With regard to hypnotics, a method of the inventioninvolves optionally measuring the binding affinity of a compound atGABA_(A) receptors having Ro15-1788 binding sites, and measuring theefficacy and EC₅₀ values of a compound at cloned α₂β₃γ₂ and α₃β₃γ₂receptors and comparing these values with the activities and EC₅₀ valuesof the compound at cloned GABA_(A) receptors containing the α₁ or α₅subunits. Optionally, the ability of the compound to mediate hypnoticeffects is assessed in vivo using an animal model established to bepredictive of sedation, and whether the compound causes cognitiveimpairment may also be assessed in vivo by an animal model shown to bepredictive of this effect.

[0042] Accordingly, this invention provides a method for identifyingcompounds with hypnotic activity that do not display the side effects ofcognitive impairment, ataxia, potentiation of alcohol effects, and atendency for tolerance and drug dependence or that display these sideeffects only at a very low level. This method comprises:

[0043] a) screening compounds, optionally measuring the binding affinityof the compounds at GABA_(A) receptors;

[0044] b) measuring the EC₅₀ and in vitro efficacy values of thecompounds at cloned α₂β₃γ₂ and α₃β₃γ₂ receptors and comparing thesevalues to the EC₅₀ and in vitro efficacy values of the compounds thatcontain the α₁ or α₅ subunit; and

[0045] c) selecting compounds with sufficiently low EC₅₀ values atα₂β₃γ₂ and α₃β₃γ₂ receptors, partial agonist activity at α₂β₃γ₂receptors and stronger partial agonist activity at α₃β₃γ₂ receptors,that also display lower activity at GABA_(A) subtype receptors thatcontain the α₁ or α₅ subunit as having hypnotic activity.

[0046] Alternatively, this assay may be performed without measuring thebinding affinity of the compound or with the additional step ofassessing the ability of the compound to mediate hypnotic effects invivo without causing cognitive impairment via an animal modelestablished to be predictive of sedation and an animal model predictiveof cognitive impairment.

[0047] Thus, the invention provides methods for identifying compoundswith hypnotic activity (1) by examining the binding of a given compoundat GABA_(A) receptors and (2) by assessing the ability of the compoundto potentiate GABA responses at a series of GABA_(A) subtype receptors.The resulting values are then compared to a set of empirically definedcriteria termed the “Hypnotic Activity Range Profiler” (Table II,below). The criteria given by the Hypnotic Activity Range Profiler areused to identify compounds with sedative hypnotic activity that have noor very minimal side effects. This activity profile comprisesmeasurements of in vitro efficacy (agonist, inverse agonist orantagonist character) and EC₅₀ values at each of 4 GABA_(A) subtypereceptors. The activity profile needed for hypnotic effects is presentedas a precise window of agonism and EC₅₀ criteria at each of thesesubtype receptors. This well-defined activity profile requires partialagonist activity at subtype receptors containing the α₂ and α₃ subunitsand lower agonist activity at other subtype receptors. The combinationof determining efficacy and EC₅₀ of a test compound is crucial as manycompounds bind with high affinity at the benzodiazepine site withoutpotentiating the GABA response at the appropriate subtype receptors.

[0048] In certain embodiments, the conclusions drawn from the in vitrodeterminations may be confirmed by examining the in vivo efficacy oftest compounds predicted to have hypnotic properties using animal modelsfor evaluating sedative activity. It may also be desirable to verifythat compounds identified by these methods possess the favorable sideeffect profiles by examining the performance of the compounds in animalmodels known to be predictive of these side effects. Thus, the animalmodels may be used as an additional step of the assay to further refinethe selection of compounds with selective hypnotic activity.

[0049] Additional Disclosure:

[0050] In a further aspect of the present invention, a method ofproviding pharmaceutical compounds to patients in need of cognitionenhancement, hypnosis, anxiolysis, and/or antidepressant treatment (suchpatients including humans, pets, livestock, and other animals) isprovided. In accordance with this method, compounds are obtained thathave been identified as having anxiolytic activity, hypnotic activity,antidepressant activity or cognition enhancing activity in accordancewith any of the novel screening, characterization, analysis oridentification methods of the present invention. Preferably suchscreening, characterization, analysis or identification is carried outoutside of the United States of America. Once such compounds have beenobtained, they are tested, preferably in vivo, for toxicity andpharmacokinetic properties. At least one compound determined to haveminimal toxic effects and to have useful pharmacokinetic properties isthen selected for clinical development. By useful pharmacokineticproperties is meant pharmacokinetic properties known in the art to beuseful for a compound having the particular activity of anxiolyticactivity, hypnotic activity, antidepressant activity or cognitionenhancing activity, as identified for each particular compound inaccordance with any of the novel screening, characterization, analysisor identification methods of the present invention for each compound. Byclinical development is meant those activities, including testing inpatients, related to the development and submission of information undera United States Federal law which regulates the manufacture, use, orsale of drugs or veterinary products, such as the Federal Food Drug andCosmetic Act and other applicable government laws and regulationspertaining thereto. The final step in this method is the offer for sale(preferably in the United States of America) for use as a drug orveterinary product of a pharmaceutical preparation (such as a pill,powder, inhalant, elixir, injectible solution, patch or suppository)comprising the compound.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The invention provides methods for screening compounds foractivity as antidepressants, cognitive enhancers, sedative hypnotics, oranxiolytics. Therapeutic compounds identified by these methods mediateeffects through the benzodiazepine site of the GABA_(A) receptor withouteliciting side effects classically associated with compounds exhibitingsuch activity that act at this site.

[0052] Although optional, it is preferred that part b) of each methodembodiment (as set forth below) will be conducted on compoundsdisplaying sufficiently potent binding affinities as determined in parta) of the method.

[0053] In one aspect of each embodiment of the method, prior todetermining an in vitro efficacy value for the test compound, thebinding affinity of the compound is measured in cells expressing clonedα, β, and γ GABA_(A) receptor subunits or in a cell membrane preparationof such cells.

[0054] In another embodiment, prior to determining the in vitro efficacyvalues for the test compound, the binding affinity of the test compoundis determined in any tissue capable of expressing GABA_(A) receptorscontaining Ro15-1788 binding sites or in a cell membrane preparation ofany tissue capable of expressing GABA_(A) receptors containing Ro15-1788binding sites.

[0055] In a preferred embodiment of the method, prior to determining invitro efficacy values of the test compound, the binding affinity of thetest compound is determined in rat spinal cord tissue or in a cellmembrane preparation of rat spinal cord tissue.

[0056] In more preferred embodiments of the method, the binding affinityof a test compound is measured in rat cortex or in a cell membranepreparation of rat cortex.

[0057] In particularly preferred embodiments, the binding affinity of atest compound is determined in rat cortex or in a cell membranepreparation of rat cortex and the test compound is selected for furtherevaluation if it gives a K₁ value of <100 nM or, preferably, <50 nM or,most preferably, <30 nM.

[0058] In these embodiments, the binding affinity of a compound may befirst determined by evaluating the ability of the compound to displace aradiolabeled compound, for example Ro15-1788 (Flumazenil), known to havehigh affinity at the benzodiazepine site.

[0059] The in vitro efficacy and EC₅₀ value of the test compound may bedetermined by measuring the chloride flux at the surface of a cellexpressing the α, β, and γ subunits of the GABA_(A) receptor in responseto GABA by the two electrode voltage-clamp technique. While a variety ofcells are suitable for use herein, the preferred cells used in thistechnique are Xenopus laevis oocytes that have been injected withnon-polyadenylated CRNA coding for human derived α, β, and γ GABA_(A)subunits. The preferred form of the γ subunit is the normally expressedlong form although an alternatively spliced form may be used.

[0060] A: Cognitive Enhancers: Cognitive enhancers identified by thismethod produce inverse agonist activity at the α1β2γ2 or α5β3γ2 GABAAsubtype receptors and agonist activity at subtype receptors containingthe α2 or α3 subunits. Of the possible subunit combinations forreceptors containing α2 or α3 subunits, the most relevant are the α2β3γ2and α3β3γ2 subtype receptors. Compounds selected according to theinvention have EC₅₀ values of about 200 nM or less at the α1β2γ2 andα5β3γ2 GABA_(A) subtype receptors and EC₅₀ values preferably of 150 nMor less at these receptors.

[0061] The criteria for screening compounds for cognitive enhancingactivity are presented below. TABLE I Cognitive Enhancer Activity RangeProfiler K₁ EC₅₀/ EC₅₀/ EC₅₀/ EC₅₀/ animal behavior effects RO15-1788efficacy efficacy efficacy efficacy (positive effect/side Rat cortex atα₁β₂γ₂ at α₂β₃γ₂ at α₃β₃γ₂ at α₅β₃γ₂ effect) <30 nM <150 nM/ any*/ any*/<150 nM/ positive effect in spatial water maze or <-10% or >10% >10%<-10% step down passive avoidance/little or >+10% no effect in seizurethreshold tests, no effect in elevated plus maze model # values at theα₁β₂γ₂ or α₅β₃γ₂ subtype receptors. On the other hand, when the EC₅₀value for a compound at either the α₂β₃γ₂ or the α₃β₃γ₂ subtype receptoris more than 100 times the EC₅₀ values for # α₁β₂γ₂ α₅β₃γ₂ subtypereceptors, then <10% in vitro efficacy is acceptable.

[0062] Thus, methods of this invention comprise:

[0063] a) screening compounds, optionally ones having a binding affinityless than 100 nM or preferably less than 30 nM at any GABA_(A) receptor;

[0064] b) determining the in vitro efficacy and EC₅₀ values for thecompounds at cloned α₁β₂γ₂ and α₅β₃γ₂ receptors;

[0065] c) determining in vitro efficacy and EC₅₀ values for thecompounds at GABA_(A) subtype receptors containing the α₂ or β₃ subunit;and

[0066] d) selecting a compound having an EC₅₀ value determined in b) ofless than 200 nM or preferably less than 150 nM, an efficacy valuedetermined in b) of less than −5% (e.g., −6%, −10%, etc.) or preferablyless than −10%, and an efficacy determined in c) of greater than 5% or,preferably, greater than 10%.

[0067] In preferred embodiments after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values for thetest compound are measured at cells expressing the α₁β₂γ₂ or α₅β₃γ₂GABA_(A) receptor subunit combinations. If the test compoundexhibits >5% inverse agonist activity (<−5% efficacy), orpreferably >10% inverse agonist activity (<−10% efficacy), at either theα₁β₂γ₂ or the α₅β₃γ₂ GABA_(A) receptor and gives EC₅₀ values of <200 nM,or more preferably <150 nM, at these subunit combinations, these valuesare compared to in vitro efficacy values determined using cellsexpressing GABA_(A) subtype receptors containing α₂ or α₃ subunits.Compounds producing >5% or preferably >10% agonist activity at thesesubunits are selected as having cognitive enhancing activity.

[0068] In a preferred embodiment, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ value aredetermined for the test compound using cells expressing the α₁β₂γ₂ orα₅β₃γ₂ GABA_(A) receptor subunit combinations. If the test compoundproduces >5% inverse agonist activity (<−5% efficacy), orpreferably >10% inverse agonist activity (<−10% efficacy), at either theα₁β₂γ₂ or α₅β₃γ₂ GABA_(A) receptors and gives EC₅₀ values of <200 nM, ormore preferably <150 nM, at these subunit combinations, these efficacyvalues are compared to the in vitro efficacy values determined withcells expressing GABA_(A) subtype receptors containing α₂β₃γ₂ or α₃β₃γ₂subunits. Where the compound produces >5% or preferably >10% agonistactivity at these subunits it is selected as having cognitive enhancingactivity.

[0069] In alternative embodiments, the method includes an in vivoevaluation of the ability of the compound to mediate cognitiveenhancement without causing proconvulsant effects. This is done usinganimal models predictive of cognition enhancement and of proconvulsantactivity. Compounds that produce a statistically significant effect inan animal model predictive of cognitive enhancement are considered to becognitive enhancing. Compounds that give either a decrease in seizurethreshold of less than 25% in the presence of a seizure inducing drug orno significant effect at the p=0.05 level are identified as lackingproconvulsant activity.

[0070] In addition, the method may include an evaluation of whether thecompound produces anxiogenic effects. This is done using an animal modelpredictive of anxiogenesis. A compound that gives no statisticallysignificant effect in the animal model predictive of anxiogenesis isidentified as lacking anxiogenic activity.

[0071] In accordance with another alternative embodiment of the method,the cognitive enhancing properties of the compound are determinedwithout measuring the binding affinity of the compound but with theadditional step of measuring the ability of the compound to mediatecognitive enhancement in vivo without proconvulsant effects, via ananimal model predictive of cognition enhancement and an animal modelpredictive of proconvulsant activity.

[0072] In accordance with yet another alternative embodiment of themethod, the cognitive enhancing properties of the compound aredetermined without measuring the binding affinity of the compound butwith the additional step of measuring the ability of the compound tomediate cognitive enhancement in vivo without proconvulsant effects andwithout anxiogenic effects, via an animal model predictive of cognitionenhancement, an animal model predictive of proconvulsant activity, andan animal model predictive of anxiogenesis, respectively.

[0073] The spatial water maze and step-down passive avoidance models aresuitable models for in vivo determinations of cognition enhancement. Thebicuculline or PTZ seizure threshold tests are suitable for use in vivoto determine proconvulsant activity. The elevated plus maze model is anexample of a model that may be used in vivo to predict anxiogenicactivity.

[0074] B: Hypnotics: Hypnotic compounds identified by this methodmediate effects through the benzodiazepine site of the GABA_(A) receptoreither without eliciting the side effects classically associated withcompounds that act at this site or elicit these side effects only tovery low degree. These side effects include cognitive impairment,ataxia, potentiation of alcohol effects, and a tendency for toleranceand drug dependence. More specifically, compounds identified ashypnotics by this method show partial agonist activity at α₂β₃γ₂receptors, stronger partial agonist activity at α₃β₃γ₂ GABA_(A) subtypereceptors, and lower activity at subtype receptors containing the α₁ orα₅ subunits. Of the possible subunit combinations for receptorscontaining α₁ or α₅ subunits the most relevant are the α₁β₂γ₂ and α₅β₃γ₂subtype receptors. Additionally, compounds useful for any of theseindications must have EC₅₀ values of 200 nM or less at the α₂β₃γ₂ andα₃β₃γ₂ GABA_(A) subtype receptors and preferably should exhibit EC₅₀values of 150 nM or less at these receptors.

[0075] The criteria for selecting a compound as having hypnoticproperties are presented below in tabular form. TABLE II HypnoticActivity Range Profiler animal behavior K+HD,1 EC₅₀/ EC₅₀/ EC₅₀/ EC₅₀/effects RO15-1788 efficacy efficacy efficacy efficacy (positive effect/Rat cortex at α₁β₂γ₂ at α₂β₃γ₂ at α₃β₃γ₂ at α₅β₃γ₂ side effect) <30 nMany*/<45% <150 nM/ <150 nM/ any*/<40% positive effect in >20% >60%spontaneous locomotor activity model/ limited effect in passiveavoidance or spatial water maze model # subtype receptors. On the otherhand, when the EC₅₀ values of the compound at the α₁β₂γ₂ or α₅β₃γ₂subtype receptor are greater than 100 times greater the EC₅₀ values atthe α₂β₃γ₂ or α₃β₃γ₂ subtype receptors, then >45% in vitro efficacy forthe α₁β₂γ₂ subtype receptor or # >40% in vitro efficacy for the α₅β₃γ₂subtype receptor is acceptable.

[0076] Thus in broad aspect, the methods of this invention comprise:

[0077] a) screening compounds, optionally, selecting compounds having abinding affinity less than 100 nM at any GABA_(A) receptor;

[0078] b) determining in vitro efficacy and EC₅₀ values for thecompounds at cloned α₂β₃γ₂ and α₃β₃γ₂ receptors;

[0079] c) determining in vitro efficacy and EC₅₀ values for thecompounds at GABA_(A) subtype receptors containing the α₁ or α₅ subunit;

[0080] d) selecting a compound having an EC₅₀ value as determined in b)of less than 200 nM, or preferably less than 150 nM, and an efficacyvalue for the α₂β₃γ₂ receptor of greater than 10%, or preferably greaterthan 20%, an efficacy value for the α₃β₃γ₂ receptor of greater than 50%,or preferably greater than 60%, an efficacy value for the receptorcontaining the α₁ subunit of less than 50%, or preferably less than 45%,and an efficacy value for the receptor containing the α₁ subunit of lessthan 45%, or preferably less than 40%.

[0081] In preferred embodiments of the invention, after the bindingaffinity of the compound has been determined, the in vitro efficacy andEC₅₀ of the test compound are measured using cells expressing the α₂β₃γ₂GABA_(A) receptor subunit combination and cells expressing the α₃β₃γ₂GABA_(A) receptor subunit combination. If the test compound exhibitsEC₅₀ values of <200 nM or more preferably <150 nM at these subtypereceptors, partial agonist activity at α₂β₃γ₂ receptors and strongerpartial agonist activity at α₃β₃γ₂ receptors, these values are comparedto the in vitro efficacy and EC₅₀ values of cells expressing subtypereceptors that contain the α₁ or α₅ subunits. If the compound alsoexhibits lower activity at receptors containing α₁ or α₅ subunits, it isselected as having hypnotic properties.

[0082] In other preferred embodiments, after the binding affinity of thecompound has been determined, the in vitro efficacy and EC₅₀ values ofthe test compound are measured using cells expressing the α₂β₃γ₂GABA_(A) receptor subunit combination and cells expressing the α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compound gives EC₅₀values of <200 nM or more preferably <150 nM at these subunitcombinations, >10% or preferably >20% agonist activity at the α₂β₃γ₂subtype receptor, and >50% or preferably >60% agonist activity at theα₃β₃γ₂ subtype receptor, these values are compared to the in vitroefficacy and EC₅₀ values in cells expressing the GABA_(A) receptorscontaining α₁ or α₅ subunits. If the compound also exhibits <50% orpreferably <45% agonist activity at GABA_(A) receptor containing the α₁subunit and exhibits <45% or preferably <40% agonist activity atGABA_(A) receptor containing the α₅ subunit, it is selected as havinghypnotic properties.

[0083] In more preferred embodiments, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values of thetest compound are measured using cells expressing the α₂β₃γ₂ GABA_(A)receptor subunit combination and cells expressing the α₃β₃γ₂ GABA_(A)receptor subunit combination. If the test compound gives EC₅₀ values<200 nM or more preferably <150 nM at these subunit combinations, >10%or preferably >20% agonist activity at the α₂β₃γ₂ subtype receptor,and >50% or preferably >60% agonist activity at the α₃β₃γ₂ subtypereceptor, these values are compared to the in vitro efficacy and EC₅₀values at cells expressing α₁β₂γ₂ and α₅β₃γ₂ subtype receptors. If thecompound also exhibits <50% or preferably <45% agonist activity at theα₁β₂γ₂ subtype receptor or exhibits <45% or preferably <40% agonistactivity at the α₁β₂γ₂ subtype receptor GABA_(A) it is selected ashaving hypnotic properties.

[0084] In alternative embodiments, the method includes measuring theability of the compound to mediate hypnotic effects in vivo withoutcausing cognitive impairment. This is accomplished using an animal modelpredictive of a compound's ability to cause hypnotic effects and ananimal model developed to be predictive of cognitive impairment. Acompound that shows a statistically significant effect in the animalmodel predictive of sedation and no statistically significant effect inthe animal model predictive of cognitive impairment is identified ashaving hypnotic properties.

[0085] Suitable in vivo animal models include the spontaneous locomotormodel for predicting hypnotic effects, and the step-down passiveavoidance model or the spatial water maze model determining cognitiveimpairment.

[0086] C: Anxiolytics: Methods are provided for identifying selectiveanxiolytic compounds. Therapeutic compounds identified by these methodsmediate effects through the benzodiazepine site of the GABA_(A) receptoreither without eliciting the side effects classically associated withcompounds that act at this site or elicit these side effects only to avery low degree. These side effects include cognitive impairment,sedation, ataxia, potentiation of alcohol effects, and a tendency fortolerance and drug dependence. More specifically, compounds identifiedas selective anxiolytics by this method show agonist activity at theα₂β₃γ₂ and α₃β₃γ₂ GABA_(A) subtype receptors and lower to no agonistactivity at subtype receptors containing the α₁ or α₅ subunits. Of thepossible subunit combinations for receptors containing α₁ or α₅subunits, the most relevant are the α₁β₂γ₂ and α₅β₃γ₂ subtype receptors.Additionally, compounds useful for any of these indications must haveEC₅₀ values of 200 rM or less at the α₂β₃γ₂ and α₃β₃γ₂ GABA_(A) subtypereceptors and preferably should exhibit EC₅₀ values of 150 nM or less atthese receptors.

[0087] The criteria for selecting a compound as having anxiolyticproperties are presented below in tabular form. TABLE III AnxiolyticActivity Range Profiler animal behavior K₁ EC₅₀/ EC₅₀/ EC₅₀/ EC₅₀/effects Ro15-1788 efficacy efficacy efficacy efficacy (positiveeffect/side Rat cortex at α₁β₂γ₂ at α₂β₃γ₂ at α₃β₃γ₂ at α₅β₃γ₂ effect)<30 nM any*/<20% <150 nM/ <150 nM/ any*/<20% positive effectin >30% >30% elevated plus maze/ no effect in spontaneous locomotoractivity model # of a compound at the α₁β₂γ₂ or α₅β₃γ₂ subtype receptorsare greater than 100 times the EC₅₀ values at the α₂β₃γ₂ or α₃β₃γ₂subtype receptors, >20% in vitro efficacy is acceptable.

[0088] Thus, in a broad aspect the invention comprises:

[0089] a) screening compounds, optionally compounds having a bindingaffinity less than 100 nM at any GABA_(A) receptor;

[0090] b) measuring in vitro efficacy and EC₅₀ values for the compoundsat cloned α₂β₃γ₂ and α₃β₃γ₂ receptors;

[0091] c) measuring in vitro efficacy and EC₅₀ values for the compoundsat GABA_(A) subtype receptors containing the α₁ or α₅ subunit; and

[0092] d) selecting a compound having an EC₅₀ as measured in b) of lessthan 200 nM and an efficacy value as measured in b) is greater than theefficacy values measured in c.

[0093] In preferred embodiments, after the binding affinity of thecompound has been determined, the in vitro efficacy and EC₅₀ of the testcompound are measured at cells expressing the α₂β₃γ₂ and α₃β₃γ₂ GABA_(A)receptor subunit combinations. If the test compound exhibits any agonistactivity and gives EC₅₀ values <200 nM, or more preferably <150 nM, atthese subunit combinations, these values are compared to the in vitroefficacies at cells expressing GABA_(A) subtype receptors containing α₁or α₅ subunits. If the compound exhibits lower or no activity at theselatter subunits, it is identified as having anxiolytic properties.

[0094] In other preferred embodiments, after the binding affinity of thecompound has been determined, the in vitro efficacy and EC₅₀ of the testcompound are measured using cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compound exhibitsany agonist activity and gives EC₅₀ values of <200 nM, or morepreferably <150 nM, at these subunit combinations, these values arecompared to the in vitro efficacy in cells expressing the α₁β₂γ₂GABA_(A) receptor subunit combination. If the compound exhibits lower tono activity at this subunit it is identified as having non-sedatinganxiolytic properties.

[0095] In further embodiments, after the binding affinity of thecompound has been determined, the in vitro efficacy and EC₅₀ of the testcompound are measured using cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compound exhibitsagonist activity and gives EC₅₀ values of <200 nM, or more preferably<150 nM, at these subunit combinations, the efficacy values are comparedto in vitro efficacy values using cells expressing the α₁β₂γ₂ and α₅β₃γ₂GABA_(A) receptor subunit combinations. If the compound exhibits lowerto no activity at these subunits it is identified as having non-sedatinganxiolytic properties.

[0096] In still other embodiments, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values for thetest compound are measured in cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compound gives >30%potentiation (i.e., increase) of the GABA response and EC₅₀ values of<200 nM, or more preferably <150 nM at these subunit combinations, theefficacy values are compared to the in vitro efficacies determined usingcells expressing GABA_(A) subtype receptors containing α₁ or α₅subunits. If the compound exhibits lower to no activity (efficacy) atthese subunits it is identified as having non-sedating anxiolyticproperties.

[0097] In yet other embodiments, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values for thetest compound are measured using cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compoundproduces >30% potentiation of the GABA response and EC₅₀ values of <200nM, or more preferably <150 nM, at these subunit combinations, theseefficacy values are compared to the in vitro efficacy values determinedusing cells expressing the α₁β₂γ₂ GABA_(A) receptor subunit combination.If the compound exhibits lower to no activity efficacy at this subunit,it is identified as having non-sedating anxiolytic properties.

[0098] In more preferred embodiments, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values for thetest compound are measured using cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compoundproduces >30% potentiation of the GABA response and EC₅₀ values of <200nM, or more preferably <150 nM, at these subunit combinations, thesevalues are compared to the in vitro efficacy values measured with cellsexpressing the α₁β₂γ₂ and α₅β₃γ₂ GABA_(A) receptor subunit combinations.If the compound produces lower to no efficacy activity at thesesubunits, it is identified as having non-sedating anxiolytic properties.

[0099] In particularly preferred embodiments, after the binding affinityof the compound has been determined, in vitro efficacy and EC₅₀ valuesfor the test compound are measured using cells expressing the α₂β₃γ₂ andα₃β₃γ₂ GABA_(A) receptor subunit combinations. If the test compoundproduces >30% potentiation of the GABA response and EC₅₀ values of <150nM at these subunit combinations, the efficacy values are compared to invitro efficacy measured using cells expressing the α₁β₂γ₂ and α₅β₃γ₂GABA_(A) receptor subunit combinations. If the compound gives <20%potentiation of the GABA response at these latter subunits, it isidentified as having non-sedating anxiolytic properties.

[0100] In alternative embodiments, the method includes the additionalstep of measuring the ability of the compound to mediate anxiolyticeffects in vivo without causing sedation. This is accomplished usinganimal models established to be predictive of anxiety and sedativeeffects. A compound that shows a statistically significant effect in theanimal model predictive of anxiety and no statistically significanteffect in the animal model predictive of sedative effects is identifiedas having non-sedating anxiolytic properties.

[0101] Suitable in viva animal models include the elevated plus mazemodel for predicting anxiolytic activity and the spontaneous locomotoractivity model to determine sedative effects.

[0102] Antidepressants: A method is provided for identifyingantidepressant compounds. Therapeutic compounds identified by thismethod act through the benzodiazepine site of the GABA_(A) receptorwithout eliciting the side effects classically associated with compoundsthat bind at this site. Alternatively, these compounds elicit the sideeffects only to a very low degree. These side effects include cognitiveimpairment, sedation, ataxia, potentiation of alcohol effects, and atendency for tolerance and drug dependence. More specifically, compoundsidentified as antidepressants by this method show agonist activity atthe α₂β₃γ₂ and α₃β₃γ₂ GABA_(A) subtype receptors and lower or no agonistactivity at subtype receptors containing the α₁ or α₅ subunits. Of thepossible subunit combinations for receptors containing α₁ or α₅subunits, preferred for use herein are the α₁β₂γ₂ and α₅β₃γ₂ subtypereceptors. Compounds useful for any of these indications must produceEC₅₀ values of 200 nM or less at the α₂β₃γ₂ and α₃β₃γ₂ GABA_(A) subtypereceptors and preferably should exhibit EC₅₀ values of 150 nM or less atthese receptors.

[0103] The preferred criteria for screening for compounds havingantidepressant activity are presented in the table below. TABLE IVAntidepressant Activity Range Profiler K₁ EC₅₀/ EC₅₀/ EC₅₀/ EC₅₀/ animalbehavior effects RO15-1788 efficacy efficacy efficacy efficacy (positiveeffect/side Rat cortex at α₁β₂γ2 at α₂β₃γ₂ at α₃β₃γ₂ at α₅β₃γ₂ effect)<30 nM any*/<20% <150 nM/ <150 nM/ any*/<20% positive effectPorsolt >30% >30% swim test/no effect in spontaneous locomotor activitymodel # for the compound at either the α₂β₂γ₂ or the α₅β₃γ₂ subtypereceptor is greater than 100 times the EC₅₀ values for either the α₂β₃γ₂or the α₃β₃γ₂ subtype receptors, then >20% in vitro efficacy isacceptable.

[0104] Thus, the method of this invention comprises:

[0105] a) screening compounds, optionally compounds having a bindingaffinity less than 100 nM at any GABA_(A) receptor;

[0106] b) determining in vitro efficacy and EC₅₀ values for thecompounds at cloned α₂β₃γ₂ and α₃β₃γ₂ receptors;

[0107] c) determining in vitro efficacy and EC₅₀ values for thecompounds at GABA_(A) subtype receptors containing an α₁ or α₅ subunit;and

[0108] d) selecting a compound having an EC₅₀ as determined in b) ofless than 200 nM and an efficacy value as determined in b) greater thanthe efficacy value measured in c.

[0109] In preferred embodiments, after the binding affinity of thecompound has been determined, in vitro efficacy and EC₅₀ values for thetest compound are measured using cells expressing the α₂β₃γ₂ and α₃β₃γ₂GABA_(A) receptor subunit combinations. If the test compound exhibitsany agonist activity and produces EC₅₀ values of <200 nM or, morepreferably, <150 nM at these subunit combinations these values arecompared to in vitro efficacy values determined using cells expressingGABA_(A) subtype receptors containing α₁ or α₅ subunits. Compoundsgiving lower or no efficacy activity at the α₁ or α₅ subunits areselected as having antidepressant properties.

[0110] In a preferred aspect, the in vitro efficacy and EC,( values aredetermined for the test compound using α₂β₃γ₂ and α₃β₃γ₂ GABA_(A)receptor subunit combinations. If the test compound exhibits agonistactivity and EC₅₀ values <200 nM or more preferably <150 nM at thesesubunit combinations, these values are compared to in vitro efficacyvalues determined with cells expressing the α₁β₂γ₂ GABA_(A) receptorsubunit combination. Where the compound exhibits lower or no efficacyactivity at the α₁β₂γ₂ subunit, it is identified as havingantidepressant properties.

[0111] In more preferred aspect, the in vitro efficacy and EC₅₀ valuesdetermined for the test compound using cells expressing the α₂β₃γ₂ andα₃β₃γ₂ GABA_(A) receptor subunit combinations are compared to in vitroefficacy values determined using cells expressing the α₁β₂γ₂ and α₅β₃γ₂GABA_(A) receptor subunit combinations combination. Where the compoundexhibits lower to no efficacy activity at both the α₁β₂γ₂ and α₅β₃γ₂subunits, it is identified as having antidepressant properties.

[0112] In these embodiments, agonist activity is preferably defined asproducing an efficacy value of >30% potentiation of the GABA response.Thus, in preferred embodiments of the invention if the test compoundexhibits >30% potentiation of the GABA response at the α₂β₃γ₂ and α₃β₃γ₂receptor subunits, and EC₅₀ values of <200 nM or, more preferably <150nM, at these subunit combinations, these values are compared to the invitro efficacy at cells expressing the α₁β₂γ₂ GABA_(A) receptor subunitcombination. Where the compound exhibits lower to no efficacy (agonist)activity at the α₁β₂γ₂ subunit, it is identified as havingantidepressant properties.

[0113] Particularly preferred embodiments comprise: determining thebinding affinity of the compound for GABA_(A) receptors determining invitro efficacy and EC₅₀ values for the test compound with cellsexpressing the α₂β₃γ₂ and α₃β₃γ₂ GABA_(A) receptor subunit combinations.Where the test compound produces >30% potentiation of the GABA response(i.e., agonist activity) and EC₅₀ values of <200 nM or, more preferably<150 nM, at these subunit combinations, these efficacy values arecompared to in vitro efficacy values determined using cells expressingthe a α₁β₂γ₂ and α₅β₃γ₂ GABA_(A) receptor subunit combinations. Wherethe compound exhibits <20% potentiation of the GABA response at theα₁β₂γ₂ and α₅β₃γ₂ subunits, it is identified as having antidepressantproperties.

[0114] In alternative embodiments, the method includes, an in vivoevaluation of the ability of the compound to mediate antidepressanteffects without causing sedation. This is done using animal modelspredictive of antidepressant activity and sedation. A compound thatproduces a statistically significant effect in an animal modelpredictive of antidepressant activity and no statistically significanteffect in an animal model predictive of sedative effects is identifiedas having antidepressant properties.

[0115] Suitable in vivo animal models include the Porsolt swim test forpredicting antidepressant activity and the spontaneous locomotoractivity model for determining sedative effects.

[0116] In the forgoing embodiments and in the claims, any of the variouscriteria presented for characterizing each of hypnotic, antidepressant,anxiolytic or cognition enhancing properties (including those set forthin the various dependent claims submitted herewith regarding aparticular one of such properties) may be applied in association witheach aspect or embodiment of the invention concerning thecharacterization of that particular property and to each of the variousindependent claims submitted herewith regarding the characterization ofthat particular property.

[0117] The methods of the present invention are illustrated further bythe following examples, which are not to be construed as limiting theinvention in scope or spirit to the specific procedures and compoundsdescribed in them.

EXAMPLES Example 1

[0118] Binding Assays

[0119] A preferred method for discovery of compounds that bind with highaffinity to GABA_(A) receptors is a competition binding assay. Ratcortex membranes are prepared by homogenizing one or two previouslyfrozen rat cortexes in 30 ml of 25 mM Tris Buffer, pH 7.4. Thehomogenate is centrifuged for 10 minutes at 500×g. The supernatant isthen transferred to a clean centrifuge tube and the pellet discarded.The supernatant is spun for 20 minutes at 48,000×g. The supernatant fromthis spin is discarded and the pellet is resuspended in 30 ml of 25 mMTris Buffer, pH 7.4 and centrifuged for an additional 10 minutes at48,000×g. The supernatant from the final spin is discarded and themembrane pellet is resuspended in 100 mL Tris buffer per gram of cortexused.

[0120] The cortex membrane preparation is used to perform either percentinhibition or competition binding assays. In order to determine percentinhibition 300 μl of resuspended membranes are mixed with 200 μl ³Hlabeled Ro15-1788 (final concentration 2.5 nM) and incubated for 1 houron ice in the presence of 2 μl test compound in DMSO (finalconcentration 4 μM) . Membranes are harvested onto untreated filtermats.The filtermats are dried and the ³H Ro15-1788 signal is counted in ascintillation counter. Nonspecific binding is determined by displacementof ³H Ro15-1788 with 10 μM Diazepam (RBI) or any other compound known tobind tightly at the benzodiazepine site.

[0121] For any test compound exhibiting a favorable percent inhibition acompetition binding curve is obtained and a K₁ value is calculated.Generally, up to 11 points spanning the compound concentration rangefrom 10⁻¹² M to 10⁻⁵ M are obtained per curve by the same method as forthe percent inhibition assay. K₁ values are calculated according to theCheng-Prussof equation. Those compounds that exhibit Ki values in thedesired range are submitted for efficacy testing.

Example 2

[0122] Electrophysiology Assays

[0123] The efficacy profile of compounds of this invention is determinedby the following electrophysiological assay for GABA_(A) receptoractivity.

[0124] Assays are carried out as described previously in White andGurley, 1995 and White, et al., 1995. Xenopus laevis oocytes areenzymatically isolated and injected with non-polyadenylated cRNA forhuman derived α, β and γ GABA_(A) subunits, respectively. In morepreferred embodiments cRNA for the α₁β₂γ_(2,) α₂β₃γ_(2,) α₂β₃γ_(2,) andα₅β₃γ₂ subunit combinations are injected. Only one of these subunitcombinations is injected per cell. For each subunit combination,sufficient message is injected to result in current amplitudes of >10 nAwhen 1 μM GABA is applied. Electrophysiological recordings are carriedout using the two electrode voltage-clamp technique at a membraneholding potential of −70 mV.

[0125] Compounds are evaluated against a GABA concentration that evokes<10% (EC₁₀) of the maximal evocable GABA current. Each oocyte is exposedto increasing concentrations of compound in order to evaluate aconcentration/effect relationship. Compound efficacy is expressed as apercent-change in current amplitude: 100*((Ic/I)−1), where Ic is theGABA evoked current amplitude observed in the presence of compound and Iis the GABA evoked current amplitude observed in the absence ofcompound.

[0126] Specificity of a compound for the Ro15-1788 site is determinedfollowing completion of the concentration/effect curve. After washingthe oocyte sufficiently to remove previously applied compound, theoocyte is exposed to GABA and 1 μM Ro-15-1788, followed by exposure toGABA_(A) 1 μM Ro15-1788, and compound. Percent change due to addition ofcompound is calculated as described above. Any percent change observedin the presence of Ro15-1788 is subtracted from the percent changes incurrent amplitude observed in the absence of 1 μM Ro15-1788. These netvalues are used for the calculation of average activity and EC₅₀ values.

[0127] To evaluate average activity and EC₅₀ values, theconcentration/effect data are averaged across cells and fit to thelogistic equation. Average values are reported as mean±standard error.In the preferred embodiment, anxiolytic compounds should exhibit anactivity profile of <20% agonist activity at the α₁β₂γ₂ and α₅β₃γ₂subunit combinations and >30 % agonist activity at the α₂β₃γ₂ and α₃β₃γ₂subunit combinations and EC₅₀ values <150 nM. Preferred hypnoticcompounds exhibit an activity profile of >20% agonist activity at theα₂β₃γ₂ subtype receptor, >60% agonist activity at the α₃β₃γ₂ subtypereceptor, <45% agonist activity at the α₁β₂γ₂ subtype receptor, and <40%activity at the α₅β₃γ₂ subunit combinations and EC₅₀ values of <150 nMat the α₂β₃γ₂ and α₃β₃γ₂ constructs. Preferred antidepressant compoundsexhibit an activity profile of <20% agonist activity at the α₁β₂γ₂ andα₅β₃γ₂ subunit combinations, >30 % agonist activity at the α₂β₃γ₂ andα₃β₃γ₂ subunit combinations, and EC₅₀ values of <150 nM at the α₂β₃γ₂and α₃β₃γ₂ constructs. Preferred cognitive enhancing compounds producean efficacy profile of >10% inverse agonist activity (<−10% efficacy) atthe α₁β₂γ₂ or α₅β₃γ₂ subtype receptors, >10% agonist activity at theα₂β₃γ₂ and α₃β₃γ₂ subunit combinations, and produce EC₅₀ values of <150nM at the α₁β₂γ₂ and α₅β₃γ₂ subtype receptors. The modulatory effect onthe GABA current amplitude is near maximal for all subtype receptorsstudied.

Example 3

[0128] Animal Behavior Methods

[0129] To verify that compounds identified as selective anxiolytics,hypnotics, antidepressants, or cognitive enhancers by this methodmediate these effects it is preferable to examine the performance ofsome such compounds in vivo. A series of animal models, most preferablyrat models, are employed for this purpose.

[0130] It has been established that the elevated plus maze model can beused to determine anxiolytic efficacy of compounds. Side effectsassociated with anxiolytic compounds can be ascertained from a varietyof animal models. The spontaneous locomotor activity model can be usedto determine whether test compounds cause sedation.

[0131] Spontaneous locomotor activity has been established as a measureof hypnotic effect. Among the most common side effects of currentlyprescribed sedative hypnotics are learning and memory deficits orcognitive impairment. Consequently, one of the goals of the sedativehypnotic program has been to develop a sedative hypnotic compound withminimal side effects of this nature. The step-down passive avoidance andspatial water maze paradigms can be used to determine whether testcompounds cause learning and memory deficits.

[0132] The Porsolt swim test has been established as a measure ofdetermining antidepressant activity of compounds. Side effectsassociated with antidepressant compounds can be ascertained from avariety of animal models. For example, the spontaneous locomotoractivity model may be used to determine whether test compounds causesedation.

[0133] The spatial water maze model or the step-down passive avoidancemodel may be used to measure cognitive enhancement. Proconvulsantactivity and anxiogenic effects are the side effects of greatest concernfor compounds that act as cognitive enhancers via the benzodiazepinesite. The bicuculline seizure threshold or the PTZ seizure thresholdtests may be used to determine whether cognition enhancing compounds areproconvulsant; the elevated plus maze may be used to determine whethersuch compounds are anxiogenic.

[0134] The absence of side effects for a compound that shows in vivoefficacy is indicative that the compound not only has the desiredactivity but that it is also a selective compound. A statisticallysignificant effect in the spatial water maze, step-down passiveavoidance, spontaneous locomotor activity, Porsolt swim test, or theelevated plus maze models is defined as p <0.05 using a valid parametricstatistical test. Likewise, no statistically significant effect isdefined in these models as p >0.05 using a valid parametric statisticaltest. No statistically significant effect for either of the seizurethreshold models is defined as a decrease of less than 25% in theseizure threshold in the presence of a seizure inducing drug or a resultthat is not significant at the p <0.05 level.

[0135] a. Elevated Plus Maze

[0136] The elevated plus maze model capitalizes on rats' innate fear ofopen, elevated places. The test apparatus is an elevated plus mazeconsisting of two open arms and two closed arms. A rat will naturallychose to spend more time on the closed arms of the maze than on the openarms but that if an efficacious anxiolytic compound is administered tothe rat prior to the test the amount of time the rat spends in the openarms is increased.

[0137] Test compound is administered IV in a 50% PEG (polyethyleneglycol) vehicle 5 minutes prior to the test session. A range of compounddoses is typically used and 8-10 rats are tested at each dose. The ratis placed in the center of the maze facing one of the open arms. Theanimal's locomotion is tracked over a five minute test session usingphotocells interfaced to a computer. The computer measures the number ofentries into each arm and the time spent on each arm. An anxiolyticeffect in the elevated plus maze model is defined by an increase in thepercentage of time spent on the open arms in compound versus vehicletreated animals.

[0138] b. Spontaneous Locomotor Activity

[0139] Spontaneous locomotor activity can be measured to determinesedative effects of compounds. Locomotor activity is measured in eightcomputerized Digiscan-16 Animal Activity Monitors (Omnitech Electronics,Columbus, Ohio) equipped with 48 infrared photocell emitters anddetectors. Each box is constructed of Plexiglas sides and floor.Horizontal activity is detected by a set of horizontal sensors on thefront to back walls and a second group of sensors on the side to sidewalls located 5 cm above the cage floor. Vertical activity is detectedby a third set of sensors on the side to side walls located 13.5 cmabove the cage floor. The rats are tested in the presence of white noise(62 dB) and red light (60 watt).

[0140] Test compound is administered IV in a 50% PEG vehicle 5 minutesprior to the test session. A range of compound doses is typically usedand 6 to 8 rats are tested at each dose. The animal's movement time,vertical activity, and total distance traveled are tracked over a 15minute test session. A sedative, or hypnotic, effect in the spontaneouslocomotor activity model is defined by a decrease in any two of thesethree measures relative to animals given vehicle alone.

[0141] c. Step-Down Passive Avoidance

[0142] In step-down passive avoidance a rat is placed on a platformlocated in the center of an electrified grid floor that is containedwithin a large (45 cm×45 cm×50 cm) white translucent Plexiglas®enclosure. The natural inclination of the rat is to step off theplatform and investigate its surroundings. In day one of the experimentanimals are treated with either Zolpidem, test compound in a 50% PEGvehicle, or vehicle alone and then trained to remain on the platform forat least 120 seconds. Each time the animal steps off the platform itreceives a mild foot shock of 2 mAmps×6 sec. Following each shock theanimal is removed from the box, placed in its cage for a one minuteinter-trial interval, and then returned to the platform. The latency tostep down on each trial, the number of trials taken to reach criterionduring training and the retention latency are collected.

[0143] Testing is conducted approximately 24 hrs. after training.Drug-free animals are placed on the platform in the box in which theywill have been trained and the latency to step down onto the grid flooris recorded for one trial as a measure of memory retention. The animalis allowed a maximum of 120 seconds to step down and does not receive ashock upon stepping off the platform.

[0144] d. Porsolt Forced Swim Test

[0145] The effects measured in this model have been correlated toantidepressant efficacy for drugs. The paradigm of this model is that aneffective antidepressant compound will cause a rat to make greaterattempts to escape a water-filled cylinder than a rat given vehicleonly.

[0146] Animals used in this study were non-naive male Sprague DawleyRats (SASCO, St. Louis) weighing between 280-350 grams. The testapparatus consists of 6 clear Plexiglas® cylinders 40 cm high×19 cmwide. Cylinders are filled to 18 cm with 25° C. water. Each rat isplaced in a cylinder for a 15 minute training session. Following eithersubchronic or acute dosing of either vehicle (0.5% methylcellulose) ofcompound, animals are brought back 24 hours later for a 5 minute testsession. These test session is videotaped for later scoring.

[0147] Subchronic dosing consists of administering drug three times inthe 24-hour period between training and testing. The drug isadministered 24 hrs., 5 hrs., and 1 hr. prior to the test session. Acutedosing consists of administering the drug once, 1 hour prior to the testsession. Scoring is done using a time-sampling computer program writtenin Visual Basic and run in DOS. Every five seconds, animals are rated asdemonstrating one of three behaviors: immobility, mild swim, orclimbing. These sampling score are then converted into percentages ofthe test session.

[0148] e. Spatial Water Maze

[0149] The spatial water maze has been used extensively as a test ofspatial learning and memory. Rats are trained to escape from the waterby swimming to a platform that is submerged just below the surface ofthe water. Since the platform is not visible to the animal, it has toutilize visual extra-maze cues in the area of the tank to locate theplatform.

[0150] The water maze apparatus consists of a circular tank, 119 cm indiameter and 56 cm in height, with a black interior. The tank is filledwith water approximately 23-25° C. to a height of 42 cm. Superimposedonto the tank are four quadrants, South, East, North and West. The tankis surrounded by external visual cues, which consist of a black andwhite checkered wall, a black and white striped wall, a white wall withtwo light fixtures, and a blue wall. A black circular PLEXIGLAS platformwith a black neoprene rubber top is placed in the Northeast quadrantapproximately 1-2 cm below the surface of the water. The submergedplatform is 39 cm in height and has a diameter of 11.5 cm. Training andtesting are conducted in the presence of a 60-62 dB white noise sourceand under dim light conditions (1.0 -1.2 lux). The animal's path istracked by a video camera interfaced to an automated tracking package(Videotrack, CPL Systems).

[0151] Acquisition Training: Training consists of six trials. Testcompound is administered IV in a 50% PEG vehicle 5 minutes prior to thetest session. A range of doses is typically used and 8 to 10 rats aretested at each dose. Each animal is placed on the platform in the tankfor 20 seconds prior to the first trial of acquisition training. For thefirst trial, the animal is placed in the water facing the wall of thetank at the “South” starting position. The order of training trials isSouth, East, North, and West. Each of the training trials is separatedby an inter-trial interval (ITI) of 3 minutes. Each trial ends with theanimal finding the platform or being placed onto it after 90 sec. Ratsare then given 10 seconds on the platform and removed from the maze forthe ITI. Each trial ends with the animal finding the platform or beingplaced onto it after 90 seconds. Rats are then given 10 seconds on theplatform and removed from the maze for the ITI. During the ITI, each ratis dried off with a towel and placed in a heating chamber maintained at45° C. The latency to reach the submerged platform (measured inseconds), the total distance traveled in the maze (measured in meters),the number of zone or quadrant transitions made, and the swim speed(measured in meters/sec.) on each trial are all recorded. Uponcompletion of training animals are returned to their home cages in thevivarium.

[0152] Retention Testing: Approximately 24 hours after training each ratis tested for retention on one trial. The rat is placed the “South”starting position and given 90 seconds to located the platform. Thelatency to locate the platform, total distance traveled, number of zonetransitions and swim speed are all recorded by computer.

[0153] f. Bicuculline Seizure Threshold Test:

[0154] This test provides a measure of whether the test compound isproconvulsant by measuring whether the test compound produces any changein the dose of the seizure inducing drug, bicuculline, needed to elicitseizures in rats. Test compounds must show <25% decrease in seizurethreshold or p >0.05 significance to be considered as drug candidates.

[0155] Adult male Sprague-Dawley rats (175-300 g) are weighed and placedin a rat restrainer. A Teflon® indwelling catheter is placed in one ofthe lateral tail veins and held in place with surgical tape. Thecatheter and a 3.0 cc syringe are connected by a length of PE-100 tubingfitted with Hamilton male and female adapter. Patency of catheterplacement is tested by backflow of venous blood and ready infusion ofless than 2 ml of saline solution. Animals are placed in a clear acryliccage for behavioral observation.

[0156] The test compound is prepared in either 25% or 50% polyethyleneglycol 400 (PEG) vehicle. A bicuculline (BIC) stock solution of 1.0mg/ml BIC in 25% PEG/75% lN HCl is also prepared. Immediately prior toinfusion 0.5 ml of BIC stock is diluted to 20 ml with 19.5 ml of icecold saline.

[0157] Test compound is infused 5 minutes prior to the start ofbicuculline infusion. Injection volumes do not exceed 4.0 ml/kg and aretypically 1.0 or 2.0 ml/kg. The catheter is flushed immediately with 2.0ml of saline to ensure total delivery of the drug. The catheter is thenpreloaded with dilute pontamine skye blue dye followed by some air toallow observation of the initiation of the bicuculline infusion. The BICsolution is delivered at a speed of 2.0 ml/minute by a constant speedinfusion pump. The final infusion rate of bicuculline is approximately0.05 mg/minute. The time elapsed from the start of bicuculline infusionto first myoclonic jerk (first head/neck twitch), the initiation of fullmyoclonus (writhing), and final forelimb extension is all recorded usinga digital stopwatch. Animals are subsequently euthanized. Thebicuculline seizure threshold is defined as the amount of bicucullinerequired to titrate the first myoclonic jerk. Bicuculline seizurethreshold is expressed in mg/kg as follows: $\frac{\begin{matrix}{\lbrack {{time}\quad {of}\quad {myoclonic}\quad {jerk}\quad ({minutes})} \rbrack \times} \\\lbrack {{infusion}\quad {rate}\quad ( {{mg}/{minute}} )} \rbrack\end{matrix}}{\lbrack {{weight}\quad {of}\quad {animal}\quad ({kg})} \rbrack}$

[0158] g. Pentelenetetrazol Seizure Threshold Test

[0159] This assay may be used as an alternative to the bicucullineseizure threshold test to determine whether a test compound isproconvulsant. Pentelenetetrazol (PTZ) is used to induce seizures ratherthan bicuculline. Compound is administered 5 minutes prior to infusionof PTZ as in the bicuculline seizure threshold test. PTZ (2.5 mg/ml in0.09% NaCl) is infused at a constant rate of 1.92 ml/minute with asyringe pump or a constant drug delivery rate of 4.8 mg/minute. The timeelapsed from the start of PTZ infusion to first myoclonic jerk (firsthead/neck twitch), the initiation of full myoclonus (writhing), andfinal forelimb extension is recorded using a digital stopwatch. The PTZseizure threshold is defined as the amount of infused PTZ required totitrate the first myoclonic jerk and is calculated by the samerelationship as used for the bicuculline seizure threshold. In eitherthe bicuculline or the PTZ seizure threshold test compounds shoulddecrease the seizure threshold by less than a 25% decrease or not show asignificant effect at the p <0.05 level.

Example 4

[0160] Anxiolytic Compounds

[0161] The compounds listed in Table V were tested by each of themethods described above for identifying anxiolytics compounds andcompared to the known anxiolytic Alprazolam. Alprazolam is known tocause side effects including sedation. TABLE V oocyte electrophysiology(EC₅₀ (nM)/maximum In vivo potentiation (%)) Behavioral assays^(†)Ro15-1788 spontaneous binding, elevated locomotor compound K₁ (nM)α₁β₂γ₂ α₂β₃γ₂ α₃β₃γ₂ α₅β₃γ₂ plus maze activity Alprazolam 3.3  37/327 12/333  69/774 10/206 0.0625 0.125 compound 1 3.6  6/12 20/71 35/7289/−11 0.03 NS  compound 2 10 33/18 12/40 23/39 36/−11 0.5 4 compound 34.7 107/26  108/52  133/68  186/38  0.125 NS* compound 4 14 48/23 19/6651/48 0 0.5 NS  compound 5 8.3 0  9/44 21/44 0 0.5 NS 

[0162] The structures of the above compounds are given in Table VI TABLEVI Compound Number Structure compound 1

compound 2

compound 3

compound 4

compound 5

Example 5

[0163] Hypnotic Compounds

[0164] The compounds listed in Table VII were tested by the methodsdescribed above for identifying hypnotic compounds and compared to theknown hypnotic Zolpidem. Zolpidem is known to display side effectsincluding cognitive impairment. TABLE VII Ro15-1788 oocyteelectrophysiology spontaneous step-down binding, (EC₅₀ (nM)/maximumpotentiation (%)) locomotor passive compound K₁ (nM) α₁β₂γ₂ α₂β₃γ₂α₃β₃γ₂ α₅β₃γ₂ activity* avoidance Zolpidem 48 178/263 553/3501776/882  >3 μM/<20  0.25 0.5 compound 6 11 44/25 28/72 33/95 108/42 0.25 4 compound 7 3  5/18  4/51  9/83 174/−23 0.125 NS^(†) compound 8 2414/17 39/42 37/61 40/16 0.5 NS  compound 9 7.8 74/33 184/53  331/23 ***/<10 0.5 0.5

[0165] The structures of Compounds 6-9 are shown in Table VIII. TABLEVIII Compound Number Structure compound 6

compound 7

compound 8

compound 9

Example 6

[0166] Antidepressant Compounds

[0167] The compounds shown in Table IX were tested by each of themethods described above and compared to the antidepressant compoundRo16-6028. TABLE IX oocyte electrophysiology EC₅₀ (nM)/ Ro15-1788maximum potentiation (%) spontaneous binding, subtype receptor Porsoltlocomotor compound K₁ (nM) α₁β₂γ₂ α₂β₃γ₂ α₃β₃γ₂ α₅β₃γ₂ swim test^(†)activity RO16-6028 0.48  3/40  5/37  5/76  3/64 1 0.06 compound 1 8159/12  114/41  144/63  88/15 20 NS* compound 3 10 33/18 12/40 23/39 36/−11 20 4 compound 4 4.7 107/26  108/52  133/68  186/38  20 NS 

Example 7

[0168] Cognition Enhancing Compounds

[0169] The following compounds were tested by the methods describedabove for identifying cognition enhancing compounds and compared to CGS8216, a compound that has been shown to have a positive effect in modelsof learning and memory. We have also shown CGS 8216 to be efficacious inthe step-down passive avoidance model (0.06 mg/kg minimal efficaciousdose). Additionally it is know that CGS 8216 is anxiogenic andproconvulsant. TABLE X oocyte electrophysiology R015-1788 (EC₅₀(nM)/maximum bicuculline binding, potentiation (%)) cognitive seizurecompound K₁ (nM) α₁β₂γ₂ α₂β₃γ₂ α₃β₃γ₂ α₅β₃γ₂ model^(†) threshold^(‡) CGS8216 0.2 nM 0.7/−26  2/−10  2/−8  1/−39 NS* 50% compound 10 10 0 31/2246/23 81/−27 0.25 NS compound 11 33 21/−11 96/21 177/36  847/−22 PO =10.0^(††) NS compound 12 5.4 18/15  11/63 18/82 19/−12 0.06 NS compound13** 6.3  3/−17 53/9  36/14 23/−20 0.125^(††) NS

[0170] Structures for Compounds 10-13 above are presented in Table XI.TABLE XI Compound Number Structure compound 10

compound 11

compound 12

compound 13**

[0171] The foregoing describes preferred embodiments of the presentinvention. Those of skill in the art will recognize that modificationsmay be made therein without departing from the spirit or scope of thepresent invention as set forth in the following claims, which concludethis specification.

What is claimed is:
 1. A method for screening a plurality of compoundsso as to identify at least one compound exhibiting cognitive enhancingactivity, comprising: a) determining in vitro efficacy and EC₅₀ valuesfor each compound at α₁β₂γ₂ or an α₅β₃γ₂ GABA_(A) subtype receptor; b)determining an in vitro efficacy value for each compound at a GABA_(A)receptor comprising an α₂ or α₃ subunit; and c) identifying asexhibiting cognitive enhancing activity a compound having: an EC₅₀ valuedetermined in a) of less than about 200 nM, an efficacy value determinedin a) of less than about −5%, and an efficacy value determined in b) ofgreater than about 5%.
 2. The method of claim 1 wherein the EC₅₀measured in step a) is less than 150 nM.
 3. The method of claim 2wherein the in vitro efficacy measured at said α₁β₂γ₂ GABA_(A) subtypereceptor or said α₅β₃γ₂ GABA_(A) subtype receptor is less than −10%. 4.The method of claim 3 wherein the in vitro efficacy measured at saidGABA_(A) receptor comprised of said α₂ subunit or said α₃ subunit isgreater than 10%.
 5. The method of claim 1 wherein the in vitro efficacymeasured at said α₁β₂γ₂ GABA_(A) subtype receptor or said α₅β₃γ₂GABA_(A) subtype receptor is less than −10%.
 6. The method of claim 5wherein the in vitro efficacy measured at said GABA_(A) receptorcomprised of said α₂ or said α₃ subunit is greater than 10%.
 7. Themethod of claim 1 wherein the GABA_(A) receptor comprised of said α₂subunit is an α₂β₃γ₂ GABA_(A) receptor or the GABA_(A) receptorcomprised of said α₃ subunit is an α₃β₃γ₂ GABA_(A) receptor.
 8. A methodfor screening compounds for cognitive enhancing activity, comprising: a)selecting compounds having a binding affinity less than 100 nM at anyGABA_(A) receptor; b) determining in vitro efficacy and EC₅₀ values foreach selected compound at an α₁β₂γ₂ or α₅β₃γ₂ GABA_(A) subtype receptor;c) determining in vitro efficacy and EC₅₀ values for each selectedcompound at a GABA_(A) receptor comprised of an α₂ or α₃ subunit; and d)identifying as having cognitive enhancing activity any compound havingan EC₅₀ value determined in b) of less than 200 nM and an efficacy valuemeasured in b) of less than −5%, and an efficacy value measured in c) ofgreater than 5%.
 9. A method of providing a pharmaceutical preparationto patients in need of cognition enhancing treatment comprising: a)obtaining at least one compound identified as exhibiting cognitionenhancing activity by the method of claim 1; b) testing said at leastone compound and submitting results of said testing as part ofsubmission of information under a United States Federal law whichregulates the manufacture, use, or sale of drugs or veterinary products;c) showing a pharmaceutical preparation comprising said at least onecompound to be safe for use as required by the provisions of the FederalFood Drug And Cosmetic Act; and d) offering the pharmaceuticalpreparation for sale in the United States of America for use as acognition enhancing drug or cognition enhancing veterinary product. 10.A method for screening a plurality of compounds for cognitive enhancingactivity, comprising: a) determining in vitro efficacy and EC₅₀ valuesfor each compound at α₁β₂γ₂ or α₅β₃γ₂ GABA_(A) receptors; b) determiningin vitro efficacy for each compound at a GABA_(A) receptor comprised ofan α₂ or α₃ subunit; c) determining the in vivo effect of each compoundin an animal model for measuring cognitive enhancement; d) determiningthe in vivo effects of each compound in an animal model forproconvulsant activity by measuring a seizure threshold in the presenceof a seizure inducing compound or in an animal model that predictsanxiogenic effects; and e) identifying a cognitive enhancing compound asa compound having cognitive enhancing properties when the EC₅₀ measuredin step a) is less than 200 nM and the efficacy measured in step a) isless than −5% and the efficacy measured in step b) is greater than 5%and said compound produces a statistically significant (p<0.05) positiveeffect in the animal model indicative of cognitive enhancement and saidcompound does not produce an effect in the animal model predictive ofproconvulsant activity of more than a 25% decrease in the seizurethreshold in the presence of the seizure inducing drug, or does notproduce a change that is statistically significant in said model, or thecompound does not produce a statistically significant effect in theanimal model that predicts anxiogenic effects.
 11. A method forscreening compounds for cognitive enhancing properties, comprising: a)selecting compounds having binding affinities of less than 100 nM at anyGABA_(A) receptor; b) measuring the in vitro efficacy of each compoundat an α₁β₂γ₂ or α₅β₃γ₂ GABA_(A) receptor; c) measuring the in vitroefficacy of each compound at a GABA_(A) receptor comprised of an α₂ orα₃ subunit; d) measuring the in vivo effect of each compound in ananimal model predictive of cognitive enhancement; e) measuring the invivo side effects of each compound in an animal model that predictsproconvulsant activity by measuring a seizure threshold in the presenceof a seizure inducing compound or measuring the in vivo side effects ofeach compound in an animal model that predicts anxiogenic effects; andf) identifying as a cognitive enhancing compound a particular compoundfor which the EC₅₀ measured in step b) is less than 200 nM and theefficacy measured in step b) is less than −5% and the efficacy measuredin step c) is greater than 5% and said particular compound produces astatistically significant (p<0.05) positive effect in the animal modelindicative of cognitive enhancement and said particular compound doesnot produce an effect in the animal model predictive of proconvulsantactivity of more than a 25% decrease in the seizure threshold in thepresence of the seizure inducing drug, or does not produce a change thatis statistically significant in said model, or said particular compounddoes not produce a statistically significant effect in the animal modelthat predicts anxiogenic effects.
 12. A method for screening compoundsfor hypnotic activity, comprising: a) determining EC₅₀ and in vitroefficacy of each compound at an α₂β₃γ₂ GABA_(A) subtype receptor or anα₃β₃γ₂ GABA_(A) subtype receptor; b) determining in vitro efficacy ofeach compound at a GABA_(A) receptor comprised of an α₁ or α₅ subunit;and c) selecting a compound having an EC₅₀ determined in a) of less than200 nM, an in vitro efficacy determined in a) of greater than 10% forsaid α₂β₃γ₂ GABA_(A) subtype receptor or greater than 50% for saidα₃β₃γ₂ GABA_(A) subtype receptor; and an in vitro efficacy valuedetermined in b) of less than 50% for the GABA_(A) receptor comprised ofan α₁ subunit or less than 45% for the GABA_(A) receptor comprised of anα₅ subunit.
 13. The method of claim 12 wherein the in vitro efficacyvalue measured at said α₂β₃γ₂ receptor is greater than 20% or the invitro efficacy value measured said α₃β₃γ₂ GABA_(A) receptor is greaterthan 60%.
 14. The method of claim 13 wherein the in vitro efficacy valuemeasured at the GABA_(A) receptor comprised of said α₁ subunit is lessthan 45% or the in vitro efficacy value measured at the GABA_(A)receptor comprised of said α₅ subunit is less than 40%.
 15. The methodof claim 12 wherein the in vitro efficacy value measured at the GABA_(A)receptor comprised of said al subunit is less than 45% or the in vitroefficacy value measured at the GABA_(A) receptor comprised of said α₅subunit is less than 40%.
 16. The method of claim 12 wherein the EC₅₀measured at said α₂β₃γ₂ GABA_(A) subtype receptor or at said α₃β₃γ₂GABA_(A) subtype receptor is less than 150 nM.
 17. The method of claim16 wherein the in vitro efficacy measured at said α₂β₃γ₂ GABA_(A)subtype receptor is greater than 20% or the in vitro efficacy measuredsaid α₃β₃γ₂GABA_(A) subtype receptor is greater than 60%.
 18. The methodof claim 17 wherein the in vitro efficacy measured at the GABA_(A)receptor comprised of said α₁ subunit is less than 45% or the in vitroefficacy measured at the GABA_(A) receptor comprised of said α₅ subunitis less than 40%.
 19. The method of claim 16 wherein the in vitroefficacy measured at the GABA_(A) receptor comprised of said α₁ subunitis less than 45% or the in vitro efficacy measured at the GABA_(A)receptor comprised of said α₅ subunit is less than 40%.
 20. The methodof claim 12 wherein the GABA_(A) receptor comprised of an α₁ subunit isan α₁β₂γ₂ GABA_(A) subtype receptor or the GABA_(A) receptor comprisedof an α₅ subunit is an α₅β₃γ₂ GABA_(A) subtype receptor.
 21. A methodfor screening a plurality of compounds so as to identify at least onecompound exhibiting hypnotic activity, comprising: a) selecting aplurality of compounds having a binding affinity of less than 100 nM atany GABA_(A) receptor. b) determining EC₅₀ and in vitro efficacy valuesfor each selected compound at an α₂β₃γ₂ GABA_(A) subtype receptor or atan α₃β₃γ₂ GABA_(A) subtype receptor; c) determining in vitro efficacyvalues for each selected compound at a GABA_(A) receptor comprised of anα₁ or an α₅ subunit; and d) identifying as exhibiting hypnotic activityeach selected compound having an EC₅₀ value determined in b) of lessthan 200 nM, an in vitro efficacy value measured in b) of greater than10% for said α₂β₃γ₂ GABA_(A) subtype receptor or greater than 50% forsaid α₃β₃γ₂ GABA_(A) subtype receptor, and an in vitro efficacy valuedetermined in c) of less than 50% for the GABA_(A) receptor comprised ofsaid α₁ subunit or less than 45% for the GABA_(A) receptor comprised ofsaid α₅ subunit.
 22. A method for screening a plurality of compounds soas to identify compounds exhibiting hypnotic activity, comprising: a)measuring the EC₅₀ and in vitro efficacy of each compound at an α₂β₃γ₂GABA_(A) subtype receptor or an α₃β₃γ₂ GABA_(A) subtype receptor; b)measuring the in vitro efficacy of each compound at a GABA_(A) receptorcomprised of an α₁ or α₅ subunit; and c) measuring the in vivo effect ofeach compound in an animal model indicative of hypnotic effects; d)measuring the in vivo effect of each compound in an animal modelindicative of cognitive impairment; and e) identifying a compound ashaving hypnotic activity when the EC₅₀ measured in step a) is less than200 nM, the in vitro efficacy measured in step a) is greater than 10%for said α₂β₃γ₂ GABA_(A) subtype receptor or greater than 50% for saidα₃β₃γ₂ GABA_(A) subtype receptor, and the in vitro efficacy measured instep b) is less than 50% for the GABA_(A) receptor comprised of said α₁subunit or less than 45% for the GABA_(A) receptor comprised of said α₅subunit and said compound produces a statistically significant (p<0.05)positive effect in the animal model indicative of sedation and saidcompound does not produce a statistically significant effect in theanimal model indicative of cognitive impairment.
 23. A method forscreening a plurality of compounds so as to identify at least onecompound exhibiting hypnotic activity, comprising: a) selectingcompounds having a binding affinity less than 100 nM at any GABA_(A)receptor; b) measuring the EC₅₀ and in vitro efficacy of each selectedcompound at an α₂β₃γ₂ GABA_(A) subtype receptor or an α₃β₃γ₂ GABA_(A)subtype receptor; c) measuring the in vitro efficacy of each selectedcompound at a GABA_(A) receptor comprised of an α₁ or α₅ subunit; and d)measuring the in vivo effect of each selected compound in an animalmodel indicative of sedative effects; e) measuring the in vivo effect ofeach selected compound in an animal model indicative of cognitiveimpairment; and f) identifying as having hypnotic activity each selectedcompound for which the EC₅₀ measured in step b) is less than 200 nM, thein vitro efficacy measured in step b) is greater than 10% for saidα₂β₃γ₂ GABA_(A) subtype receptor or greater than 50% for said α₃β₃γ₂GABA_(A) subtype receptor, and the in vitro efficacy measured in step c)is less than 50% for the GABA_(A) receptor comprised of said α₁ subunitor less than 45% for the GABA_(A) receptor comprised of said α₅ subunitand said compound produces a statistically significant (p<0.05) positiveeffect in the animal model indicative of hypnotic effects and saidcompound does not produce a statistically significant effect in theanimal model indicative of cognitive impairment.
 24. A method forscreening a plurality of compounds so as to identify compoundsexhibiting anxiolytic activity, comprising: a) determining in vitroefficacy and EC₅₀ value for each compound at an α₂β₃γ₂ GABA_(A) subtypereceptor or an α₃β₃γ₂ GABA_(A) subtype receptor; b) determining in vitroefficacy values for each compound at a GABA_(A) receptor comprised of anα₁ subunit or an α₅ subunit; and c) identifying as exhibiting anxiolyticactivity each compound having an EC₅₀ value determined in a) of lessthan 200 nM and an efficacy value measured in a) greater than theefficacy measured in b).
 25. The method of claim 24 wherein the EC₅₀measured in step a) is less than 150 nM.
 26. The method of claim 25wherein the in vitro efficacy measured at said α₂β₃γ₂ or said α₃β₃γ₂GABA_(A) receptor is greater than 20%.
 27. The method of claim 25wherein the in vitro efficacy measured at said α₂β₃γ₂ or said α₃β₃γ₂GABA_(A) receptor is greater is than 30%.
 28. The method of claim 27wherein the in vitro efficacy measured at said GABA_(A) receptorcomprised of said α₁ or said α₅ subunit is less than 20%.
 29. The methodof claim 24 wherein the in vitro efficacy measured at said α₂β₃γ₂ orα₃β₃γ₂ GABA_(A) receptor is greater than 20%.
 30. The method of claim 24wherein the in vitro efficacy measured at said α₂β₃γ₂ or α₃β₃γ₂ GABA_(A)receptor is greater than 30%.
 31. The method of claim 30 wherein the invitro efficacy measured at said GABA_(A) receptor comprised of said α₁or said α₅ subunit is less than 20%.
 32. The method of claim 24 whereinthe GABA_(A) receptor comprised of said α₁ subunit is an α₁β₂γ₂ GABA_(A)subtype receptor or the GABA_(A) receptor comprised of said α₅ subunitis an α₅β₃γ₂ GABA_(A) subtype receptor.
 33. A method for screening forcompounds having anxiolytic activity, comprising: a) selecting acompound having a binding affinity less than 100 niM at any GABA_(A)receptor; b) measuring in vitro efficacy and EC₅₀ values for eachcompound at an α₂β₃γ₂ or α₃β₃γ₂ GABA_(A) receptor; c) measuring in vitroefficacy values for each compound at a GABA_(A) receptor comprised of anα₁ or α₅ subunit; and d) selecting a compound having an EC₅₀ valuemeasured in a) of less than 200 nM and an efficacy value measured in b)greater than the efficacy measured in c).
 34. A method for screeningcompounds so as to select at least one compound having anxiolyticactivity, comprising: a) measuring in vitro efficacy for each compoundat an α₂β₃γ₂ GABA_(A) subtype receptor or an α₃β₃γ₂ GABA_(A) subtypereceptor; b) measuring in vitro efficacy and EC₅₀ values for eachcompound at a GABA_(A) receptor comprised of an α₁ or α₅ subunit; c)measuring in vivo effects of each compound in an animal model indicativeof anxiolytic activity; d) measuring in vivo effects of each compound inan animal model indicative of sedative effects; and e) selecting eachcompound having: an EC₅₀ value measured in a) of less than 200 nM, anefficacy value measured in b) greater than the efficacy measured in stepc), a statistically significant (p<0.05) positive effect in the animalmodel indicative of anxiolytic activity, and no statisticallysignificant effect in the animal indicative of sedative effects.
 35. Amethod for screening a plurality of compounds so as to identify at leastone compound having anxiolytic activity, comprising: a) selecting acompound having a binding affinity less than 100 nM at any GABA_(A)receptor; b) measuring in vitro efficacy and EC₅₀ values for eachselected compound at an α₂β₃γ₂ or α₃β₃γ₂ GABA_(A) receptor; c) measuringin vitro efficacy for each selected compound at a GABA_(A) receptorcomprised of an α₁ or α₅ subunit; d) measuring in vivo effects of eachselected compound in an animal model indicative of anxiolytic activity;e) measuring in vivo effect of each selected compound in an animal modelindicative of sedative effects; and f) selecting a compound having: anEC₅₀ value measured in b) of less than 200 nM, an efficacy measured inc) greater than the efficacy measured in d), a statistically significant(p<0.05) positive effect in the animal model indicative of anxiolyticactivity, and no statistically significant effect in the animalindicative of sedative effects.
 36. A method for screening a pluralityof compounds so as to identify compounds exhibiting antidepressantactivity, comprising: a) determining in vitro efficacy and EC₅₀ valuesfor each compound using an α₂β₃γ₂ GABA_(A) subtype receptor or an α₃β₃γ₂GABA_(A) subtype receptor; b) determining in vitro efficacy values foreach compound at a GABA_(A) receptor comprised of an α₁ or α₅ subunit;and c) identifying as having antidepressant activity a compound havingan EC₅₀ value determined in a) of less than 200 nM and an efficacy valuedetermined in a) of greater than the efficacy value determined in b).37. The method of claim 36 wherein the EC₅₀ value determined using saidα₂β₃γ₂ GABA_(A) subtype receptor or said α₃β₃γ₂ GABA_(A) subtypereceptor is less than 150 nM.
 38. The method of claim 37 wherein the invitro efficacy measured at said α₂β₃γ₂ or said α₃β₃γ₂ GABA_(A) receptoris greater than 20%.
 39. The method of claim 37 wherein the in vitroefficacy measured at said α₂β₃γ₂ GABA_(A) subtype receptor or saidα₃β₃γ₂ GABA_(A) subtype receptor is greater than 30%.
 40. The method ofclaim 39 wherein the in vitro efficacy measured at said GABA_(A)receptor comprised of said α₁ subunit or said α₅ subunit is less than20%.
 41. The method of claim 36 wherein the in vitro efficacy measuredat said α₂β₃γ₂ GABA_(A) subtype receptor or said α₃β₃γ₂ GABA_(A) subtypereceptor is greater than 20%.
 42. The method of claim 36 wherein the invitro efficacy measured at said α₂β₃γ₂ GABA_(A) subtype receptor or saidα₃β₃γ₂ GABA_(A) subtype receptor is greater than 30%.
 43. The method ofclaim 42 wherein the in vitro efficacy measured at said GABA_(A)receptor comprised of said α₁ subunit or said α₅ subunit is less than20%.
 44. The method of claim 36 wherein the GABA_(A) receptor comprisedof said α₁ subunit is an α₁β₂γ₂ GABA_(A) subtype receptor or theGABA_(A) receptor comprised of said α₅ subunit is an α₅β₃γ₂ GABA_(A)subtype receptor.
 45. A method for screening compounds forantidepressant activity, comprising: a) selecting compounds having abinding affinity less than 100 nM at any GABA_(A) receptor; b)determining in vitro efficacy and EC₅₀ values for the selected compoundsusing an α₂β₃γ₂ GABA_(A) subtype receptor or an α₃β₃γ₂ GABA_(A) subtypereceptor; c) determining in vitro efficacy for the selected compoundsusing a GABA_(A) receptor comprised of an α₁ or an α₅ subunit; and d)identifying as having antidepressant activity a compound having an EC₅₀as determined in b) of less than 200 nM and an efficacy value asdetermined in b) greater than the efficacy value determined in c).
 46. Amethod for screening compounds for antidepressant activity, comprising:a) determining in vitro efficacy and EC₅₀ values for each compound usingan α₂β₃γ₂ GABA_(A) subtype receptor or α₃β₃γ₂ GABA_(A) subtype receptor;b) determining in vitro efficacy values for each compound at a GABA_(A)receptor comprised of an α₁ or an α₅ subunit; c) determining in vivoeffect of said compound in an animal model indicative of antidepressantactivity; d) determining the in vivo effect of said compound in ananimal model indicative of sedative effects; and e) identifying as anantidepressant a compound that produces an EC₅₀ value as determined ina) of less than to 200 nM, and an efficacy value as determined in b)greater than the efficacy value from c), and (i) produces astatistically significant (p<0.05) positive effect in the animal modelindicative of antidepressant activity and (ii) does not produce astatistically significant effect in the animal model indicative ofsedative effects.
 47. A method for screening compounds forantidepressant activity, comprising: a) selecting test compounds havinga binding affinity less than 100 nM at any GABA_(A) receptor; b)determining in vitro efficacy and EC₅₀ value for each test compoundusing an α₂β₃γ₂ GABA_(A) subtype receptor or an α₃β₃γ₂ GABA_(A) subtypereceptor; c) determining in vitro efficacy value for each test compoundat a GABA_(A) receptor comprised of an α₁ subunit or an α₅ subunit; d)determining the in vivo effect of each test compound in an animal modelindicative of antidepressant activity; e) determining the in vivo effectof each test compound in an animal model indicative of sedative effects;and f) identifying as an antidepressant a compound that produces: anEC₅₀ value as determined in b) of less than 200 nM, an efficacy value asdetermined in c) greater than the efficacy value from d), and (i)produces a statistically significant (p<0.05) positive effect in theanimal model indicative of antidepressant activity and (ii) does notproduce a statistically significant effect in the animal modelindicative of sedative effects.
 48. A method of providing pharmaceuticalcompounds to patients in need of hypnotic treatment comprising: a)obtaining at least one compound identified as exhibiting hypnoticactivity by the method of claim 21; b) testing said at least onecompound and submitting results of said testing as part of submission ofinformation under a United States Federal law which regulates themanufacture, use, or sale of drugs or veterinary products c) showing apharmaceutical preparation comprising said at least one compound to besafe for use as required by the provisions of the Federal Food Drug AndCosmetic Act; and d) offering the pharmaceutical preparation for sale inthe United States of America for use as an hypnotic drug or hypnoticveterinary product.
 49. A method of providing a pharmaceuticalpreparation to patients in need of anxiolytic treatment comprising: a)obtaining at least one compound identified as exhibiting anxiolyticactivity by the method of claim 24; b) submitting information regardingthe anxiolytic activity of said at least one compound as part of anapplication under a United States Federal law which regulates themanufacture, use, or sale of drugs or veterinary products c) showing apharmaceutical preparation comprising said at least one compound to besafe for use as required by the provisions of the Federal Food Drug AndCosmetic Act; and d) offering the pharmaceutical preparation for sale inthe United States of America for use as an anxiolytic drug or anxiolyticveterinary product.
 50. A method of providing a pharmaceuticalpreparation to patients in need of antidepressant treatment comprising:a) obtaining at least one compound identified as exhibitingantidepressant activity by the method of claim 36; b) testing said atleast one compound and submitting results of said testing as part ofsubmission of information under a United States Federal law whichregulates the manufacture, use, or sale of drugs or veterinary productsc) showing a pharmaceutical preparation comprising said at least onecompound to be safe for use as required by the provisions of the FederalFood Drug And Cosmetic Act; and d) offering the pharmaceuticalpreparation for sale in the United States of America for use as anantidepressant drug or antidepressant veterinary product.